Accurate segregation of chromosome, initiated by abrupt and irreversible dissolution of sister-chromatid cohesion at anaphase, is crucial for the faithful inheritance of parental genomes during eukaryotic cell division. The dissolution of sister-chromatid cohesion is catalyzed by separase after the destruction of securin by the anaphase-promoting complex/cyclosome (APC/C). However, separase was localized to the mitotic centrosome, raising the question as how separase hydrolyzes sister-chromatid cohesion of centromere at the anaphase onset. Here we show that separase is associated with mitotic chromosomes and this association is regulated by Aurora B kinase. Using a panel of separase antibodies, we found that separase protein was accumulated in mitosis and degraded at the end of telophase. To study the spatiotemporal distribution of separase in mitosis, we carried out immunofluorescence microscopic analyses. Surprisingly, separase was found to be associated with mitotic chromosomes from prophase to metaphase and dissociated from the chromosomes in anaphase right after sister chromatids separation. Staining of isolated mitotic chromosomes from Nocodazole-arrested cells revealed that separase is concentrated at the centromeric cohesion. To examine if any mitotic kinases are responsible for chromosomal localization of separase in mitosis, we carried out RNAi-mediated knockdown and found that association of separase with mitotic chromosomes was a function of Aurora B. Consistent with the phenotype seen in the Aurora B-repressed cells, inhibition of Aurora B kinase by hersperadin prevents the association of separase with chromosomes. Our results suggest that Aurora B kinase activity helps coordinate the association of separase with chromosome and the initiation of sister-chromatid separation.